ObjectiveTo investigate the protective effect and the regulation mechanism of oxaloacetate (OAA) on myocardial ischemia reperfusion injury in rats. MethodsSixty rats, weight ranged from 200 to 250 grams, were randomly divided into 6 groups:a negative control group, a sham operation control group, a model control group, an OAA pretreatment myocardial ischemia-reperfusion model group (three subgroups:15 mg/kg, 60 mg/kg, 240 mg/kg). We established the model of myocardial ischemia reperfusion of rats and recorded the internal pressure of left ventricle (LVSP), the maximal rate of left ventricular pressure change (±dp/dtmax) and left ventricular end diastolic pressure (LVEDP). We restored reperfusion 180 minutes after ligating the left anterior descending coronary artery 30 minutes and determinated cardiac troponin Ⅰ (cTn-I), lactate dehydrogenase (LDH), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px). We took out heart tissues, stained it and calculated the infarcted size. We used the Western blot to detect the expression of NF-E2 related factor 2 (Nrf2), Kelch-like ECH-associated protein-1 (Keap1) and heme oxygenase-1 (HO-1). ResultsCompared with the sham operation group, heart function indexes in the negative control group had no significant difference (P>0.05). But in the model control group there was a decrease (P<0.05) And the serum levels of LDH, cTn-I, and myocardial infarcted size were significantly increased (P<0.01). Compared with the model control group, heart function indexes in the OAA pretreatment groups improved, the serum LDH, cTn-I activity, and infarct size decreased (P<0.05), SOD and GSH-Px activity increased (P<0.05). And these results were statistically different (P<0.01) in the high dose OAA pretreatment groups. Compared with the model control group, the expression of Keap1 in the OAA pretreatment group was down-regulated (P<0.001) while total Nrf2, nucleus Nrf2 and its downstream HO-1 was up-regulated (P<0.001), which suggested that OAA enhanced antioxidant capacity by (at least in part) Keap1-Nrf2 pathway, resulting in reducing myocardial damage and protecting myocardium after acute myocardial ischemia reperfusion injury. ConclusionOxaloacetate can provide protective effects on myocardial ischemia reperfusion injury through down-regulating the expression of Keap1 and up-regulating the expression of Nrf2 and its downstream peroxiredoxins to improve antioxidant capacity.
Objective To compare the positive rate of zinc finger protein A20， NF-κB p65 protein， and P-glyco- protein between primary hepatocellular carcinoma （HCC） tissues and paratumor tissues， and to explore the relationship between the 3 kinds of proteins and pathological features of HCC. Methods Thirty-two HCC tissues and 26 paratumor tissues resected from patients with HCC treated in our hospital from Feb. 2009 to Aug. 2010 were enrolled. Clinical data were also collected from files. The expressions of zinc finger protein A20， NF-κB p65 protein， and P-glycoprotein were tested by immunohistochemistry. Results The positive rate of zinc finger protein A20， NF-κB p65 protein， and P-glycoprotein in HCC tissues were 87.5%（28/32）， 81.3%（26/32）， and 65.6%（21/32）， respectively， which were higher than that in paratumor tissues〔61.5%（16/26）， 34.6%（9/26）， and 30.8%（8/26）， respectively〕， P＜0.05. The three kinds of proteins were all closely related with HbsAg， and zinc finger protein A20 was related with cirrhosis in addition （P＜0.05）. Conclusions The positive rate of zinc protein A20， NF-κB p65 protein， and P-glycoprotein are much higher in primary HCC tissues than that in paratumor tissus， and they may play an important role in preoperative determination of hepatic tumors.
Objective Lots of metal ions accumulation and over-expression of receptor activator of NF-κB l igand (RANKL) around the prosthesis could be found in revision of total hip arthroplasty. To investigate the relationship between metal ions and aseptic loosening by observing the effects of Co2+ and Cr3+ ions on the expression of RANKL and osteoprotegerin(OPG) from osteoblast. Methods Osteoblasts were cultured in vitro at the density of 1 × 105 cells/mL, and were divided into 2 groups according to different culture solutions. In control group, osteoblasts were cultured with normal medium without CoCl2 and CrCl3. In experimental group, osteoblasts were cultured with the medium including CoCl2 (10 mg/ L) and CrCl3 (150 mg/L) solutions. The RT-PCR and ELISA methods were appl ied to detect the mRNA expression of RANKL and OPG and protein level at 24 and 48 hours after co-cultured, respectively. Results RT-PCR revealed that the mRNA expression of RANKL and OPG could be found in two groups at 24 and 48 hours after co-cultured, the expression was higher in the experimental group than in control group, especially the expression of RANKL, showing significant difference (P lt; 0.05). At 24 and 48 hours after co- cultured, the ratios of RANKL mRNA to OPG mRNA in the experimental group were 0.860 and 1.232, respectively, which were significantly higher than those in the control group (0.695 and 0.688,P lt; 0.05). ELISA revealed that the protein level of RANKL and OPG in experimental group were significantly higher than those in the control group (P lt; 0.05). Conclusion Co2+ and Cr3+ can stimulate the mRNA expressions of RANKL, OPG and secretion of those protein from osteoblasts, especially increase of the RANKL, which promotes the formation and activation of osteoblasts and the generation of aseptic loosening.
Objective To observe the human mononuclear cell releasing TNF-α and the activation of Caspase-3 during apoptosis after stimulated by Co2+ and Cr3+, to discuss the mechanism of artificial joint wear production metal ion on aseptic loosening. Methods CoCl2 powder and CrCl3 powder were dissolved by asepsis inject water, preparing solution for10 mg/L and 500 mg/L, respectively. Mononuclear cells were acquired from peripheral blood, 4 × 106/culture dish. According to the difference of culture solution, the cells were divided into 3 groups. Group A: mononuclear cell was culture with normal sal ine as control; group B: mononuclear cell was cultured with CoCl2 solution; group C: mononuclear cell was cultured with CrCl3 solution. The production of TNF-α was assessed by ELISA, the activation of Caspase-3 was measured by colorimetric assay and the apoptotic cell was detected by TUNEL assays at 12, 24 and 48 hours after co-cultured respectively. Results The concentration of TNF-α and the expression of Caspase-3 in groups B and C were higher than those in group A (P lt; 0.05), and reached the peak level at 24, 48 hours, respetively. The TUNEL positive cells were detected in the all groups, nucleus was pyknotic and darker-staining, cell body was crinkle and cell membrane was integrity. There were significant differences in the apoptosis rate between groups B, C and group A (P lt; 0.05). And the activation of Caspase-3 increased and had the positive correlation with the apoptosis rate (r=0.765). Conclusion Co2+ and Cr3+ ions can stimulate human mononuclear cell to release TNF-α and induce human mononuclear cell apoptosis, which result in periprosethetic osteolysis and related to activation of Caspase-3.
Objective To investigate the influence of diammonium glycyrrhizinate (DG) on the expression of NF-κB and neuron apoptosis after spinal cord ischemia-reperfusion injury in rats. Methods Fourty-eight healthy SD male rats, weighing 220-270 g, were randomly divided into the experimental group and the control group, with 24 rats in each group. A model of spinal cord ischemia-reperfusion injury was completed by intercepting the rats’ abdominal aorta between right and left renal arteries for 30 minunts. In the experimental group, each rat was injected 20 mg/kg DG via subl ingual vein 10 minutes before ischemia occurred. Equal qual ities of physiological sal ine were injected into the rats in the control group. The two groups were observed at 3, 24, 72 and 168 hours after ischemia-reperfusion, respectively. Lumbar myeloid tissues were prepared at the different times, respectively. The expression of NF-κB p65 in lumbar myeloidtissues was analyzed by immunohistochemistry and the apoptosis of neurons was examined by TUNEL reaction. Meanwhile, histological changes of spinal cord were observed by HE staining. Then the correlation between NF-κB and neuron apoptosis was analyzed. Results HE staining showed obvious histological changes of spinal cord of the two groups. In the control group, myeloid tissue edema and normal neurons were observed at 3 hours; there were more histological changes at 24 hours and 72 hours; vacuolus in gray matters and some survived neurons were seen at 168 hours. The histological changes at each time in the experimental group were fewer than those in the control group. The immunohistochemical staining showed that the expression of NF-κB p65 was observed. After ischemia-reperfusion, the expression strengthened at 3 hours, reached the peak at 24 hours and then weakened slowly. At 3, 24, 72 and 168 hours after ischemia-reperfusion, the absorbency (A) value of NF-κB p65 in the experimental group was 0.306 0 ± 0.024 4, 0.396 4 ± 0.022 7, 0.296 6 ± 0.021 1 and 0.267 9 ± 0.015 3, respectively, and that in the control group was 0.361 1 ± 0.017 7, 0.496 6 ± 0.020 1, 0.356 3 ± 0.021 0 and 0.301 4 ± 0.018 1, respectively. There were significant differences between the two groups (P lt; 0.05). The inhabitation ratio of NF- κB p65 expression by DG was 15.40%, 20.17%, 19.28% and 11.11% at 3, 24, 72 and 168 hours after ischemia-reperfusion, respectively. Neuron apoptosis was observed, which strengthened at 3 hours and was the most serious at 24 and 168 hours after ischemia-reperfusion. At 3, 24, 72 and 168 hours after ischemia-reperfusion, the A value of neuron apoptosis in the experimental group was 0.171 0 ± 0.029 1, 0.175 5 ± 0.031 1, 0.175 1 ± 0.027 9 and 0.183 2 ± 0.023 7, respectively, and that in the control group was 0.236 8 ± 0.063 6, 0.241 2 ± 0.042 6, 0.201 5 ± 0.049 8 and 0.250 1 ± 0.048 4, respectively. There were significant differences between the two groups (P lt; 0.05). The inhabitation ratio of neuron apoptosis by DG was 27.79%, 27.23%, 13.08% and 26.74% at 3, 24, 72 and 168 hours after ischemia-reperfusion, respectively. The expression of NF-κB in myeloid tissues was positively correlated with neurons apoptosis in the two groups (r = 0.838, P lt; 0.01). Conclusion Spinal cord ischemia-reperfusion injury may cause a marked expression of NF-κB and notable evidence of neurons apoptosis. DGcan reduce neurons apoptosis by inhibiting the expression of NF-κB.
OBJECTIVE: To determine the influence of basic fibroblast growth factor (bFGF) on endothelial cell (EC) proliferation in vitro and its possible mechanisms, and to examine the effect of both TNP-470 and dexamethasone (Dex) on the EC proliferation induced by bFGF. METHODS: Human umbilical vein endothelial cells were cultured and the proliferation of EC was quantified by a colorimetric assay using MTT reagent. The expression of nuclear factor-kappa B (NF-kappa B) and ki-67 was detected with SABC immunohistochemical method. RESULTS: bFGF stimulated the EC proliferation and enhanced the expression of NF-kappa B and ki-67 in nucleus; TNP-470 and Dex suppressed EC proliferation induced by bFGF, and reduced the expression of NF-kappa B and ki-67 in nucleus. CONCLUSION: The above results indicate that the possible mechanisms of EC proliferation stimulated by bFGF come from that bFGF can activate NF-kappa B to promote the synthesis of DNA and EC mitosis. TNP-470 and Dex inhibited EC proliferation stimulated by bFGF by inhibiting NF-kappa B.
Objective To study the effect of advanced glycosylation end products (AGEs) on human retinal pigment epithelium (RPE) cells. Methods Human primary RPE cells were cultured in basal and different concentrations of AGEs with different times. The cells were divided into several groups as follows: group C (control): bovine serum albumin 0.1 g/L, 24 hours (C1) and 48 hours (C2); group NC (normal control): basal culture medium with 5.6 mmol/L of glucose, 24 hours (NC1) and 48 hours (NC2); group A (AGEs): 0.1 g/L, 24 hours and 48 hours, A1 and A4; 0.2 g/L, 24 hours and 48 hours, A2 and A5; 0.4 g/L, 24 hours and 48 hours, A3 and A6. Immunohistochemistry analysis was used to study the protein expression of receptor for AGEs (RAGE), peroxisome proliferativeactivated receptor-gamma coactivator-1 alpha (PCG -1α) and vascular endothelial growth factor (VEGF) protein. The activation of nuclear factor-kappa B (NF-κB) was detected by confocal microscope. Software IPP6.0 and SPSS 17.0 were used to analyze the quantitation data. Results Immunohistochemistry analysis showed that RAGE protein, PGC-1α protein and VEGF protein were basally secreted in RPE cells, but AGEs can obviously increases the expression level of these proteins (F=294.5, 228.3, 241.5; P＜0.05). Confocal microscope demonstrated that AGEs increased the activation of NF-κB significantly. Conclusion Accumulation of AGEs can stimulate the expression of RAGE protein, PGC-1α protein and VEGF protein, activation of NF-κB and induce apoptosis of RPE cells.
Objective To investigate the effects of knocking down Rac1 gene (ras-related C3 botulinum toxin substrate 1) by small hairpin RNA (shRNA) on retinal neovascularization in a mouse model of oxygen-induced retinopathy (OIR). Methods One hundred and eight 7-day-old C57BL/6J mice were divided into three groups randomly.The OIR was induced by Smith protocol in 2 groups. OIR mice received an intravitreal injection of Rac1-shRNA plasmid or the nonsense plasmid in the geneintervention group and control group respectively at the age of postnatal day 11 (P11). Non-OIR mice also received an intravitreal injection of Rac1-shRNA plasmid at P11 as the blankintervention group which lived in the normoxic environment.Retinal neovascularization was investigated on flat-mounts after fluorescence angiography at P15 and P17. Endothelial cell nuclei breaking through the internal limiting membrane were counted on pathological section at P17.The expression of Rac1 and NF-kappa;B p65 subunit was measured by immuohistochemistry, Western blot, real-time polymerase chain reaction (RT-PCR) and in situ hybridization. Results Compared with the blank-control group,the level of Rac1 mRNA in the gene-intervention group decreased obviously（t=4.500，P=0.001）;the retinal non-perfusion areas,fluorescence leakage, neovascularization and the number of endothelial cell nuclei breaking through the internal limiting membrane were reduced significantly（t=6.521,P＜0.001）; the level of NF-kappa;B p65 nuclear translocation decreased（t=16.008,P＜0.001）while the expression of NF-kappa;B p65 mRNA was reduced obviously（t=3.354,P=0.006）, which was positively correlated with the expression of Rac1-mRNA (P=0.012).Conclusion Intravitreal injection of Rac1-shRNA with liposome in mice can effectively inhibit the expression of Rac1,and inhibit the retinal neovascularization under relative hypoxia via blocking the ROS-NF-kappa;B pathway.
Objective To investigate the expression of nuclear factor (NF)-κB and intercellular adhesion molecule (ICAM)-1 in rat′s retina injured by ischemia-reperfusion, and the effect of pyrrolidine dithiocarbamate (PDTC) on the expression of NF-κB and ICAM-1. Method The model of retinal ischemia-reperfusion was set up in 60 SD rats, which were divided into two groups with 30 rats in each: ischemia-reperfusion group and ischemia-reperfussion with injection of PDTC group. The left cephalic artery of each rat was ligated, and the right side was the control. Every group was subdivided into group 1 hour, 6, 12, 24, 48, and 72 hours after ischemia-reperfusion injury, and with 5 rats in each group. mRNA of NF-κB and ICAM-1 mRNA was measured by in situ hybridization (ISH) method in rat′s retina. Every rat underwent electroretinography (ERG) at the corresponding time before executed by neck breaking. Results In ischemia-reperfusion group, expression of NF-κB and ICAM-1 was detected at the 6th hour after ischemia-reperfusion, reached the highest level at the 24th hour, and weakened gradually later. In ischemia-reperfusion with injection of PDTC group, expression of NF-κB and ICAM-1 was detected at the 12th hour after ischemia-reperfusion, and reached the highest level at the 24th hour but lower than that in ischemia-reperfusion group. No expression of NF-κB and ICAM-1 was found in the control group. The relative recovery rate of ERG a and b wave amplitude in ischemia-reperfusion groups was lower than that in ischemia-reperfusion with injection of PDTC group at every stage(P＜0.01 ). The lowest relative recovery rate of ERG a and b wave amplitude in different stages in both of the 2 groups was at the 24th hour(P＜0.01). Conclusions NF-κB and ICAM-1 may play an important role in retinal ischemia-reperfusion injury, as the inhibitor of NF-κB, PDTC may relieve the retinal ischemia-reperfusion injury. (Chin J Ocul Fundus Dis，2004，20：175-178)
Objective To detect expression of NF-κB in the inner retina and in vestigate the inhibitoryeffect of pyrrolidine dithiocarbamate on retinal neovascularization in rats. Methods The rat models with retinopathy were set up un der the hypoxia condition, and fluorescein fundus angiography (FFA) was used to observe the retinal neovascularization. The expressions of NF-κB in the inner retina in rats with and without neovascularization were detected by immunohisto chemical method. PDTC was intraperitoneally injected in rats with neovascularization to observe the expression of NF-κB in the inner retina and the effect on retinal neovascularization. Results Hypoxia induced NF-κB activation in the retinal glial cells and endothelial cells. But immuno-staining intensity for NF-κB and adhesion molecules were reduced by PDTC intraperitoneal injection. Retin al angiogenesis in rats were suppressed effectively (P＜0.05). Conclusions NF-κB activation correlates with retinal neovascularization closely. PDTC may inhibit the NF-κB activation and prove beneficial in the treatment of ischemic neovascularization. (Chin J Ocul Fundus Dis，2003，19：201-268)